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Am J Physiol Renal Physiol 293: F624-F630, 2007. First published June 13, 2007; doi:10.1152/ajprenal.00398.2006
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Met-RANTES reduces endothelial progenitor cell homing to activated (glomerular) endothelium in vitro and in vivo

Maarten B. Rookmaaker,1 Marianne C. Verhaar,1 Hetty C. de Boer,2 Roel Goldschmeding,3 Jaap A. Joles,4 Hein A. Koomans,4 Hermann-Josef Gröne,5 and Ton J. Rabelink2

1Department of Vascular Medicine, University Medical Center Utrecht, Utrecht; 2Department of Nephrology, Leiden University Medical Center, Leiden; Departments of 3Pathology and of 4Nephrology and Hypertension, University Medical Center Utrecht, Utrecht; and 5Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany

Submitted 7 October 2006 ; accepted in final form 31 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The chemokine RANTES (regulated upon activation normal T-cell expressed and secreted) is involved in the formation of an inflammatory infiltrate during glomerulonephritis. However, RANTES receptor inhibition, although reducing glomerular leukocyte infiltration, can also increase damage. We hypothesized that RANTES does not only promote the influx and activation of inflammatory leukocytes but also mediates glomerular microvascular repair by stimulating the homing of bone marrow (BM)-derived endothelial progenitor cells. To investigate the role of RANTES in the participation of BM-derived cells in glomerular vascular repair, we used a rat BM transplantation model in combination with reversible anti-Thy-1.1 glomerulonephritis. Twenty-four hours after the induction of glomerulonephritis, BM-transplanted rats were treated for 7 days with either the RANTES receptor antagonist Met-RANTES or saline. The participation of BM-derived endothelial cells in glomerular repair, glomerular monocyte infiltration, and proteinuria was evaluated at days 7 and 28. Furthermore, we used an in vitro perfusion chamber assay to study the role of RANTES receptors in shear-resistant adhesion of the CD34+ stem cells to activated endothelium under flow. In our reversible glomerulonephritis model, RANTES receptor inhibition specifically reduced the participation of BM-derived cells in glomerular vascular repair by more than 40% at day 7 without impairing monocyte influx. However, no obvious change in recovery from proteinuria or morphological damage was observed. Blockade of RANTES receptors on CD34+ cells in vitro partially inhibited platelet-enhanced, shear-resistant firm adhesion of the CD34+ cells to activated endothelium. In conclusion, our data suggest that RANTES is involved in the homing and participation of BM-derived endothelial cells in glomerular repair.

regulated upon activation normal T-cell expressed and secreted; glomerulonephritis; regeneration

GLOMERULAR INFLAMMATION plays an important role in the pathogenesis of various renal diseases, including glomerulonephritides, vasculitides, and renal transplant rejection. Chemotactic cytokines (chemokines) are involved in many stages of the inflammatory response: they mediate leukocyte recruitment and activation during initiation, as well as progression of inflammation. Anti-chemokine strategies are currently being developed for various potential clinical indications. The chemokine RANTES (regulated upon activation normal T-cell expressed and secreted; CCL5) has been proposed as a promising target for therapeutic intervention in renal disease (3, 16, 36). RANTES is a member of the CC-chemokine family and a ligand for a number of chemokine receptors, including CCR1, CCR3, and CCR5. Inhibition of the RANTES receptor CCR5 was shown to be associated with decreased inflammatory infiltrate and reduced inflammatory reaction in several glomerulonephritis models (36). In renal transplant rejection models, blockade of RANTES receptors reduced the inflammatory reaction and improved acute and chronic allograft rejection (16, 54). However, it was recently reported that RANTES receptor antagonists may also have unfavorable effects that may limit the clinical applicability of such a strategy. In horse apoferritin-induced glomerulonephritis, RANTES inhibition, although reducing glomerular leukocyte infiltration, aggravated glomerular damage (2). Furthermore, increased glomerular damage in RANTES receptor CCR1 knockout mice during nephrotoxic glomerulonephritis has been reported (57). In both models, the aggravation included an important increase in vascular injury. Thus RANTES may not only promote the influx and activation of leukocytes but may also be involved in the repair response of the renal vasculature.

Evidence is accumulating that circulating bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to renal repair (21, 40, 44, 45). The mechanisms underlying recruitment and homing of these progenitor cells to glomerular endothelium have not been fully elucidated but may involve several chemokines. Interestingly, RANTES receptors (CCR3, CCR5, and CCR9) were shown to be present on BM-derived stem cells (23, 47, 63). In addition, RANTES was reported to be involved in vascular formation (6, 34, 43). We hypothesize that RANTES mediates glomerular microvascular repair by stimulating the homing of EPC and that inhibition of RANTES receptors results in decreased homing and participation of EPC, reducing the repair response. We used a rat BM transplantation (BMT) model to assess whether the participation of BM-derived endothelial cells in glomerular repair is RANTES dependent in vivo. An in vitro flow model was used to investigate whether RANTES is involved in the adhesion of stem cells to activated endothelial cells.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Animal model. Male 11-wk-old WAG/RijHsd (RT-1Au) (WR) and Brown Norway/RijHsd (RT-1An) (BN) rats, weighing 200–250 g, were purchased from Harlan (Horst, The Netherlands). The animals were kept in filter-top cages and received sterilized food and acidified water ad libitum. The ethics committee of the University Medical Center Utrecht approved all protocols. Allogenic BMT was performed in BN rats with WR rats as BM donors to generate rat BM-chimeras (WRBM->BN rats), as described previously (44, 61). Flow cytometry of peripheral blood samples showed that at 4 wk after BMT, 87 ± 3% of the circulating leukocytes in the recipient BN rats were of WR origin (range 76–91%; no significant difference between groups; P = 0.65).

Experimental design. The monoclonal antibody (mAb) anti-rat Thy-1.1 (ER4, mouse IgG2a, 1 mg/kg body wt) was used to induce reversible mesangiolytic glomerulonephritis. To investigate the role of the chemokine RANTES, daily tail-vein injections with the receptor antagonist Met-RANTES were applied (200 µg/day, kindly provided by Amanda E. Proudfoot, Serono Pharmaceutical Research Institute, Geneva, Switzerland). Met-RANTES is formed by the addition of a single methionine residue to the amino terminus of RANTES, which turns it into a potent RANTES receptor antagonist (41). Met-RANTES infusions were not started until 24 h after the induction of glomerulonephritis to prevent interference with the formation of an inflammatory infiltrate that peaks at 4 h after antibody infusion (30). The experimental groups were as follows: group 1, anti-Thy-1.1 (1 mg/kg) at day 0 and saline starting at day 1 for 7 days (n = 8); group 2, anti-Thy-1.1 at day 0 and Met-RANTES starting at day 1 for 7 days (n = 8); group 3, saline infusion at day 0 and Met-RANTES starting at day 1 for 7 days (n = 4); group 4, control group (n = 4) with only saline infusions. Rats were killed at day 7 or day 28 after induction of glomerular injury. One rat was excluded from group 1 on day 28 because anti-Thy-1.1 was extravasated during the injection procedure. At the end of the experimental protocol, rats were anesthetized with pentobarbital sodium (intraperitoneal injection of 60 mg/kg) and exsanguinated, and the kidneys were perfused in situ at 120 mmHg with 4°C PBS for 3 min to remove circulating cells from the renal vasculature. Kidney specimens were cut transversely into three slices. Two parts were embedded in Tissue-Tek OCT compound (Sakura Finetek Europe BV, Zoeterwoude, the Netherlands) and snap-frozen in liquid nitrogen for immunofluorescence. One slice was fixed in 4% buffered formalin and embedded in paraffin for morphological studies.

Renal morphology and function. Renal morphology was evaluated in periodic acid-Schiff-stained sections (3 µm) by normal light microscopy (Leitz Laborlux 12; Leitz, Wetzlar, Germany). A blinded observer evaluated all slides. Urine was collected for determination of urinary protein at days –1, 2, 5, 9, 13, 20, and 27. Rats were weighed and placed in metabolic cages with free access to food and water. Twenty-four-hour urinary protein loss was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories). Plasma and urinary creatinine levels were determined colorimetrically (Sigma Diagnostics, St. Louis, MO). Creatinine clearance, calculated by a standard formula, was used as an estimate of glomerular filtration rate.

Immunofluorescence double staining. To identify the cell type of BM-derived glomerular cells, immunofluorescent double staining was performed on 5-µm cryostat sections as previously described (44). The mAb U9F4, directed against major histocompatibility complex (MHC)-I expressed on WR and not BN cells, was used to detect donor-derived cells (55). U9F4 was used in combination with the following primary antibodies to identify donor-derived endothelial cells and monocytes or macrophages: rat endothelial cell antigen-1 (RECA-1), a murine IgG1 mAb against a surface antigen presented on all rat endothelial cells (12) (Serotec, Oxford, UK); and ED-1, a murine IgG1 mAb to a cytoplasmic antigen present in monocytes and macrophages (kindly provided by Ed Dub, Dept. of Cell Biology, Free University, Amsterdam, the Netherlands). We used a biotin-streptavidin detection system to prevent cross reactivity of the secondary antibodies with the anti-rat Thy-1.1 mAb ER4. The antibodies U9F4, RECA-1, and ED-1 were biotinylated using the biotinylation reagent in the DAKO Animal Research Kit for mouse primary antibodies (DAKO, Glostrup, Denmark). In short, after blocking endogenous biotin (biotin blocking kit, Vector Laboratories, Burlingame, CA), acetone-fixed sections were incubated with the biotinylated primary antibody (ED-1 or U9F4) followed by incubation with fluorescein isothiocyanate (FITC)-conjugated streptavidin (fluorescent streptavidin kit, Vector Laboratories). The signal was augmented with goat anti-streptavidin-FITC (Vector Laboratories). After blocking the remaining biotin binding sites (biotin blocking kit, Vector Laboratories), we incubated the sections with the second biotinylated primary antibody (U9F4 or RECA-1) followed by Texas red isothiocyanate (TRITC)-conjugated streptavidin (fluorescent streptavidin kit, Vector Laboratories). To exclude the possibility that secondary antibodies were picking up the wrong primary, controls were included in which the procedure was performed with substitution of the first primary antibody with a different biotinylated control antibody or with substitution of the second primary antibody with a different biotinylated control antibody. All control stainings were negative, indicating that the biotin blocking steps were complete and that no cross-reactivity has occurred. Sections were numbered and evaluated by two blinded investigators. To assess the number of BM-derived endothelial cells and monocytes in the glomeruli, the amount of cells in 20 glomeruli was averaged. To quantify the amount of preserved resident endothelial cells after anti-Thy-1.1 nephritis, the RECA-1/U9F4 double staining was combined with 4,6-diamino-2-phenylindole (DAPI; Roche, Almere, The Netherlands) nuclear staining to facilitate the identification of individual endothelial cells. RECA-1-positive, U9F4-negative cells containing a DAPI-positive nucleus were considered preserved resident endothelial cells. Cells were counted using a Leica DMR microscope (Leica, Wetzlar, Germany) at a 630-fold magnification. Cells were identified as BM-derived endothelial cells or monocytes/macrophages based on immunofluorescent double staining with U9F4/RECA-1 or U9F4/ED-1, respectively, as well as morphological characteristics.

Quantitative RT-PCR. Total RNA was extracted from 30 mg frozen renal cortex using RNeasy columns (Qiagen, Valencia, CA). After cDNA synthesis, expression of VEGF-A mRNA was assessed by quantitative real-time PCR using TaqMan Gene Expression Assays with predesigned probe and primers (Applied Biosystems, Foster City, CA). TATA-box binding protein was used as internal reference.

Isolation of CD34+ cells from cord blood. CD34+ cells were isolated from human umbilical cord blood (UCB). The CD34+ cell fraction from UCB has previously been shown to contain cells that can differentiate into endothelial cells (32, 46). UCB was recovered in special collection bags containing citrate, phosphate, and dextrose (Maco Pharma, Tourcoing, France) immediately after delivery. Protocols for sampling UCB were approved by the institutional ethical committee. UCB collection was performed with written approval of the parents before labor and delivery. The mononuclear cell fraction was isolated by gradient separation on Ficoll (Amersham, Hertogenbosch, the Netherlands). CD34+ cell isolation was performed on an automated magnetic cell sorter (Automacs, Miltenyi Biotech, Bergisch Gladbach, Germany) using the direct CD34 isolation kit (Miltenyi Biotech) according to the instructions of the manufacturer. Purity of the cells was for ~95% as assessed by fluorescence activated cell sorter (FACS) analysis (Becton Dickinson, Erembodegem, Belgium) using an anti CD34-FITC labeled antibody (Becton Dickinson). CD34+ cells were kept at 4°C at a stock concentration of 107/ml in endothelial growth medium-2 (EGM-2, Biowhittaker/Cambrex, Verviers, Belgium) without additional growth factors and were used within 1 h after isolation.

Isolation of platelet-rich plasma from peripheral blood. Platelet-rich plasma (PRP) was prepared by centrifugation (15 min at 150 g at room temperature, no brake) of low-molecular-weight heparin (LMWH, Pharmacia, Woerden, the Netherlands) anticoagulated whole blood from an aspirin-free healthy donor. PRP was kept at room temperature until use.

Microvascular endothelial cell culture. Human microvascular endothelial cells (HMEC), an immortalized cell line originally isolated from human foreskin, were obtained from the Centers for Disease Control and Prevention (Atlanta, GA) (1). Cells were cultured on gelatin-coated glass coverslips (18 x 18 mm, Laboroptik, Marienfeld, Germany) in EGM-2 supplemented with growth factors and cytokines (bullet kit, Biowhittaker). At confluence, cells were stimulated for 6 h with tumor necrosis factor-{alpha} (TNF-{alpha}; 10 ng/ml, Boehringer, Ingelheim, Germany) to induce tissue factor (TF) expression. After 6 h, the TNF-{alpha} was washed away, and the HMEC were kept in EGM-2 medium until use.

CD34+ cell perfusion and evaluation. Perfusion chamber assays were performed under steady-flow conditions as previously described (9). In short, the CD34+ cell suspension (1 x 106/ml) was aspirated through the perfusion chamber containing a coverslip with HMEC. The flow chamber was mounted on a microscope stage (DM RXE, Leica) equipped with a black-and-white charge-coupled device video camera (Sanyo, Osaka, Japan). Platelets facilitate the initial tethering and rolling of mononuclear cells under flow and provide the RANTES that has been shown to stimulate leukocyte adhesion (49, 59). De Boer et al. (9) recently demonstrated that platelets enhance the adhesion of CD34+ cells to activated endothelial cells by more than 15 times. Therefore, HMEC were pretreated as follows. Platelet aggregates were formed on a monolayer of TF-expressing HMEC by perfusing LMWH-PRP for 5 min at 1 dyn/cm2 (flow rate 80 µl/min) at 37°C. The platelet layer was rinsed for 5 min at 1 dyn/cm2 with HEPES buffer.

CD34+ perfusions were performed as individual runs under specific shear conditions (flow at 80 µl/min; shear stress at 1 dyn/cm2; 5 min at 37°C). Shear stress was then increased to 2.0 dyn/cm2, and recording of the images on VHS video recorder (Panasonic) was started. Video images of 13 different frames were evaluated for the number of CD34+ cells in contact with the monolayer of HMEC. The surface area of the thrombi was measured using a computer-assisted program (Scion Image) and expressed in arbitrary units (AU) per frame; the number of HMEC-adherent CD34+ cells per frame was counted, and the ratio of HMEC-adherent CD34+ cells per surface area of thrombi was calculated. The contribution of RANTES in shear-resistant binding to endothelial cells was studied by preincubation of the CD34+ cells with Met-RANTES (1 µg/ml for 15 min at 37°C) (26, 39, 41). Met-RANTES was kept present during the perfusion.

Statistics. All values are expressed as means ± SD. Statistical significance (defined as P < 0.05) was evaluated using the Student's t-test or ANOVA with Bonferroni post hoc tests where appropriate. Proteinuria in time was assessed using a multivariate analysis.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Met-RANTES treatment reduces the influx of glomerular BM-derived endothelial cells in experimental glomerulonephritis. Reversible glomerular damage was induced by intravenous injection of anti-Thy-1.1 antibodies. To assess the role of RANTES in glomerular repair, RANTES receptors were inhibited using daily Met-RANTES injections, starting 24 h after the induction of glomerulonephritis to prevent interference with the formation of an inflammatory infiltrate and subsequent glomerular damage (30). Participation of BM-derived endothelial cells in glomerular repair after induction of glomerulonephritis was assessed using immunofluorescent double staining in WRBM->BN rats. Cells staining positive both with BM donor-specific antibodies (U9F4) (Fig. 1A) and endothelial specific antibodies (RECA-1) (Fig. 1B) were identified as BM-derived endothelial cells (Fig. 1C). One week after anti-Thy-1.1 injection there was a significant increase in BM-derived endothelial cells per glomerular section (6.8 ± 2.0 vs. 1.0 ± 0.4; P < 0.01). The number of BM-derived endothelial cells in peritubular capillaries and vasa recta was very limited and not influenced by anti-Thy-1.1 injection (data not shown). In the Met-RANTES-treated nephritic group, there was also a significant increase in BM-derived endothelial cells per glomerular section (3.9 ± 1.6 vs. 1.0 ± 0.4; P < 0.05) 1 wk after anti-Thy-1.1 injection (Fig. 1D). However, this increase was significantly reduced compared with the non-Met-RANTES-treated group (P < 0.05). Met-RANTES treatment did not influence the amount of preserved resident endothelial cells per glomerular section 7 days after induction of anti-Thy-1.1 nephritis (P = 0.67). Although BM-derived cells stimulate neovascularization by growth factor secretion, no difference in VEGF-A mRNA expression could be demonstrated in the renal cortex (P = 0.64). The number of BM-derived endothelial cells per glomerular section appears to decrease between day 7 and day 28 in both the Met-RANTES-treated and nontreated nephritic rats (3.1 ± 0.8 and 3.3 ± 0.5, respectively)(Fig. 1D). In nonnephritic control rats, Met-RANTES treatment did not influence the number of BM-derived endothelial cells per glomerular section (1.4 ± 0.4 vs. 1.0 ± 0.4; P = 1.00) (Fig. 1D).


Figure 1
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  		Fig. 1. Bone marrow (BM)-derived endothelial cells in the glomerulus. Immunofluorescent staining of glomerular sections from WRBM->BN rats at 7 days after induction of anti-Thy-1.1 nephritis (A–C; x630 magnification). [WRBM->BN rats are rat BM chimeras generated from allogenic BM transplantation performed in Brown Norway/RijHsd (RT-1An) (BN) rats with WAG/RijHsd (RT-1Au) (WR) rats as BM donors.] A: staining for BM donor (WR) major histocompatibility complex-I (U9F4, green). B: staining for endothelial cells [rat endothelial cell antigen-1 (RECA-1), red]. C: U9F4/RECA-1 double staining identifies BM-derived endothelial cells (U9F4/RECA-1 double-positive, yellow; arrows). D: average number of BM-derived endothelial cells per glomerular section 7 and 28 days after anti-Thy-1.1 injection with (filled bars) or without Met-RANTES treatment (open bars). After 7-day treatment with Met-RANTES, no increase in BM-derived endothelial cells is observed. Seven days after anti-Thy-1.1 injection, there is a significant increase in BM-derived endothelial cells in both the group treated with and without Met-RANTES. This increase is significantly reduced in the Met-RANTES-treated group. After 28 days, there is a significant decrease in BM-derived endothelial cells in the group not treated with Met-RANTES. *Significant increase compared with control population. **Significant decrease compared with day 7. §Significant difference in BM-derived endothelial cells per glomerular section between the groups treated with and without Met-RANTES. RANTES, regulated upon activation normal T-cell expressed and secreted.

 
Met-RANTES treatment does not influence the influx of monocytes/macrophages in experimental glomerulonephritis. Injection of anti-Thy-1.1 in WRBM->BN chimeric rats induced an inflammatory reaction. Inflammatory infiltration was quantified by immunofluorescent double staining with monocyte-specific antibodies (ED-1) and BM donor MHC-I-specific antibodies (U9F4). Cells that stained positive for both U9F4 and ED-1 were identified as BM-derived monocytes. A significant increase in glomerular BM-derived monocytes was seen at day 7 in nephritic rats (13.9 ± 1.4 vs. 1.4 ± 0.5; P < 0.01). This amount decreased to 11.0 ± 1.0 at day 28. Almost all glomerular monocytes were of donor BM origin (>95%). Seven-day treatment with Met-RANTES did not significantly influence the influx of monocytes at 7 or 28 days after anti-Thy-1.1 injection (10.9 ± 3.3; P = 0.31 and 7.6 ± 1.3; P = 0.23). In nonnephritic control rats, Met-RANTES treatment did not influence the number of BM-derived monocytes per glomerular section (1.6 ± 0.4 vs. 1.4 ± 0.5; P = 1.00).

Met-RANTES treatment does not influence anti-Thy-1.1-induced glomerular damage. Histologic analysis showed acute mesangiolysis, capillary ballooning, and microaneurysm formation at day 7. The number of glomerular sections containing a microaneurysm increased from 3.0 ± 1.8% to 36 ± 3% at 7 days after anti-Thy-1.1 injection (P < 0.01). Creatinine clearance decreased from 1.81 ± 0.29 ml/min at day 0 to 1.60 ± 0.35 ml/min at day 7. Proteinuria increased from 17 ± 5 mg/24 h at day 0 to a maximum of 90 ± 25 mg/24 h followed by a gradual decline to 63 ± 16 mg/24 h at day 28. Met-RANTES treatment neither influenced microaneurysm formation (24 ± 11% at day 7; P = 0.13) nor the decrease in creatinine clearance (1.25 ± 0.39 ml/min at day 7; P = 0.10). Proteinuria was not significantly influenced by treatment with Met-RANTES either in quantity (peak: 98 ± 46 mg/24 h; day 28: 84 ± 62 mg/24 h; P = 0.90) or in development over time (P = 0.80). In control (not anti-Thy-1.1 treated) rats, no significant influence of Met-RANTES treatment was observed at day 7 on either glomerular microaneurysm formation (7.8% ± 3.1%; P = 1.00), creatinine clearance (1.20 ± 0.42 ml/min; P = 0.10), or urinary protein excretion (17.5 ± 5 mg/24 h; P = 0.54).

Blockade of RANTES receptors partially inhibits shear-resistant, firm adhesion of CD34+ cells to activated endothelium. TF-expressing HMEC were preperfused for 5 min at 1 dyn/cm2 with LMWH-anticoagulated PRP. In time, irregularly shaped thrombi were formed, consistent with our previous observations (9). Platelet aggregate formation was more pronounced at the inlet of the perfusion chamber. CD34+ cells tethered on these thrombi, detached, and attached firmly to the endothelial monolayer downstream of the thrombus (Fig. 2A). CD34+ cells that adhered to HMEC were counted, and the surface area of thrombi was measured using a computer-assisted program (Fig. 2B). Figure 2D shows that in the absence of a thrombus (e.g., at the outlet of the perfusion chamber), only a few CD34+ cells adhered to the TNF-{alpha}-stimulated HMEC. An increase in surface area of the thrombi showed increasing numbers of endothelial cell (EC)-adherent CD34+ cells per frame.


Figure 2
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  		Fig. 2. Attachment of CD34+ stem cells to activated human microvascular endothelial cells (HMEC) after preperfusion with platelet-rich plasma. Flow direction is from right to left in A, B, and C, respectively. CD34+ stem cells attach downstream from the thrombi in absence (A and B) and presence (C) of Met-RANTES. Adhesion of CD34+ cells and thrombus surface area were analyzed by computer (B and C). The number of adhering bright, round HMEC-adherent CD34+ stem cells was counted and the thrombus surface area measured (B: open arrowhead, example of adhering cell; arrow, example of thrombus surface area). D and E show correlation between the number of adhering CD34+ cells and thrombus surface area in absence ({circ}, solid line in D; no Met-RANTES in E) and presence of Met-RANTES (bullet, dashed line in D; Met-Rantes in E).

 
Platelet aggregation elaborates chemokines such as RANTES, which has been shown to be involved in the shear-resistant adhesion of monocytic cells to inflamed endothelial cells (49). A possible involvement of the RANTES/CC chemokine receptor pathway in the firm adhesion of CD34+ cells to endothelial cells was investigated by preincubation of CD34+ cells with Met-RANTES (1 µg/ml), which was kept present during the perfusion. A range of 0.01–1 µg/ml has previously been shown to completely block RANTES-mediated shear resistant adhesion of monocytes on IL-1beta-activated microvascular endothelial cells (16). Blockade of RANTES-receptors on CD34+ cells with Met-RANTES partially inhibited shear-resistant, firm adhesion of the CD34+ cells to the endothelial cells, located downstream of the platelet thrombi (Fig. 2C). The ratios of EC-adherent CD34+ cells per thrombus surface area were calculated (Fig. 2E) in the absence and presence of Met-RANTES. Inhibition of the RANTES/CCR pathway showed a significant decrease in binding of endothelial cell-adherent CD34+ cells calculated per thrombus area (2.05 ± 0.69 vs. 0.58 ± 0.27; P < 0.001).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The chemokine RANTES is known for its role in the homing and activation of inflammatory cells (4, 14, 15). Blockade of RANTES receptors has been proposed as potential therapeutic strategy in glomerulonephritis. However, there is increasing evidence that chemokines not only increase inflammation and subsequent tissue damage but also regulate the inflammatory response, preventing excessive damage (18, 60, 65). Our study suggests that RANTES receptors are involved in participation of EPC in endothelial and glomerular repair. This may limit the use of RANTES receptor antagonists to attenuate inflammatory damage.

Previously, we (44) have shown in a rat BMT model that BM-derived EPC participate in glomerular repair. A single injection with the antibody to the Thy-1 antigen causes acute mesangial cell loss, matrix dissolution, and loss of endothelial cells in this model, with maximal injury at 4–7 days, followed by a repair phase (7, 13, 22, 53). In glomeruli of chimeric rats, we identified a small number of donor BM-derived endothelial and mesangial cells, which increased significantly after induction of anti-Thy-1.1 glomerulonephritis. Here, we demonstrated that treatment of the chimeric rats with Met-RANTES from day 1 until day 7 after induction of anti-Thy-1.1 glomerulonephritis causes a marked reduction in the number of BM-derived endothelial cells in glomeruli by more than 40% at day 7 after anti-Thy-1 injection, without affecting the number of preserved resident endothelial cells per glomerular section. These observations suggest that the influx and incorporation of BM-derived endothelial cells in glomerular repair is RANTES dependent. In our experiment, Met-RANTES treatment was started 1 day after induction of glomerular injury to prevent interference with the initial inflammatory response. The inflammatory infiltrate is formed within 4 h and is already resolving after 24 h (30). Indeed, in our experiments the glomerular influx of monocytes was not significantly different between Met-RANTES-treated and control animals.

Interestingly, despite the marked reduction of BM-derived endothelial cells in the glomeruli, no significant difference was observed in functional and morphological recovery. An explanation for the absence of a significant functional difference in our model could be that homing and incorporation of BM-derived EPC has no relevance to glomerular repair. However, recent reports have demonstrated that infused BM-derived cells incorporate into the endothelial lining and reduce endothelial injury and mesangial activation in anti-Thy-1.1-induced glomerulonephritis (25, 58). Redundant or "back-up" repair mechanisms may be present in the glomerulus: local proliferation of mature glomerular endothelial cells on the one hand, and homing and incorporation of circulating BM-derived progenitor cells on the other (20, 22, 29, 35, 44). In our healthy animals with relatively mild anti-Thy-1.1-induced injury, local repair mechanisms may have been appropriate for the damage inflicted; however, with increasing injury or with impaired local regeneration mechanisms, as occurs in disease states associated with endothelial dysfunction, the role of circulating progenitor cells may become crucial for recovery (48). Alternatively, undifferentiated BM-derived cells that do not integrate in the vasculature, and are therefore less dependent on shear stress resistant cell adhesion, might stimulate glomerular repair by paracrine stimulation of local endothelial cells. Finally, the lack of functional and morphological consequences could be related to the relatively short period of Met-RANTES treatment.

The marked reduction in the number of BM-derived endothelial cells in injured glomeruli after Met-RANTES treatment suggests that RANTES is involved in the homing of EPC and their incorporation into injured endothelium. Platelet aggregation elaborates chemokines such as RANTES. Platelets can enhance neovascularization (8, 37, 38, 42) and have recently been demonstrated to stimulate the homing and differentiation of EPC (9, 24). Using a perfusion chamber assay, we demonstrated that blockade of RANTES receptors on CD34+ mononuclear cells with Met-RANTES partially inhibited platelet-enhanced, shear-resistant firm adhesion of the CD34+ cells to activated immortalized human microvascular endothelium, indicating that platelets augment neovascularization at least partially by RANTES-enhanced CD34+ cell adhesion. The renal fenestrated endothelium is unique and quite distinct from other types of endothelium; however, previous studies have demonstrated that the RANTES receptors are also expressed in glomerular endothelial cells in vivo (3). Our findings suggest that RANTES receptors may be directly involved in CD34+ adhesion to activated glomerular endothelial cells; however, also other mechanisms can be proposed. The chemokine receptor CXCR4 and its ligand SDF-1 have been shown to be involved in the homing of CD34+. Recent reports demonstrated that RANTES receptor stimulation can modulate the stimulation of CXCR4 by SDF-1, which is secreted by activated platelets (10, 19, 28, 52, 64). The CD34+ hematopoietic stem cell population has been shown to contain cells that can differentiate into endothelial cells (5, 11). Myeloid cells also play an important role in progenitor cell-mediated neovascularization. It has been suggested that immature myeloid cells are an important source of EPC (27, 51). Furthermore, Grunewald et al. (17) demonstrated that homing of myeloid cells is an early event in adult neovascularization and that these pioneering myeloid cells produce factors, like SDF-1, that attract EPC (31, 33, 56). Met-RANTES may indirectly inhibit homing and participation of EPC by reducing homing of myeloid cells (16). However, in our glomerulonephritis model, Met-RANTES treatment did not reduce the number of ED-1+ myeloid cells in the glomerulus. Other mechanisms may contribute to the effects of Met-RANTES on the participation of EPC in glomerular repair. Chemokines like RANTES have been shown to be involved in the regulation of cell proliferation and apoptosis (50, 62). Met-RANTES may reduce progenitor cell differentiation and/or reduce the survival of EPC, thereby also reducing the number of EPC in the injured glomeruli.

Taken together, in addition to the well-known role of RANTES in the induction and modulation of inflammation, our data suggest that RANTES stimulates the homing and participation of EPC in renal vascular regeneration. In response to glomerular injury, the role of RANTES appears to shift from inflammatory toward angiogenic. Our findings offer an explanation for the differences in response to RANTES inhibition in glomerulonephritis models. The changing role of the chemokine RANTES during inflammation, from proinflammatory to proangiogenic, stresses the importance of better understanding of these processes. This may lead to better therapeutic strategies in renal disease, for example, by more adequate timing or combining of chemokine-directed therapies.


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 ABSTRACT
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This work was financially supported by Dutch Kidney Foundation Grant PC 127 and Netherlands Heart Foundation Grant NHS-2002B157. M. C. Verhaar is supported by NWO VENI Grant 016.036.041.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Verhaar, Dept. of Vascular Medicine, F02.126, Univ. Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (e-mail: m.c.verhaar{at}umcutrecht.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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